Over the past decade, the use of herbal remedies has skyrocketed in popularity among North Americans. Such remedies are complex mixtures of plant constituents, which are believed to interact synergistically to elicit biological responses. However, very little is known about the specific mechanisms of synergy involved in the biological activities of plant medicines. The objective of the proposed research is to investigate synergistic interactions among the chemical constituents of Echinacea and Spilanthes, two plants with purported immune-enhancing activities. This research is significant because it is expected to provide mechanistic information about the immunological activities of two medicines that are widely employed throughout the US and Europe as immune enhancers. The rationale for carrying out these studies is that an understanding of the mechanisms by which Spilanthes and Echinacea extracts interact with the immune system is expected to aid in the development of novel strategies for the use of plant medicines to treat or prevent diseases with immune related causes. The specific aims of this study are to determine which constituents from Spilanthes have immunomodulatory activity, and to assess the potential that these constituents interact synergistically with each other and with those of Echinacea to modulate immune activity. In carrying out this research, the hypothesis will be tested that alkylamides from Spilanthes interact synergistically with each other and with other constituent groups from Spilanthes and Echinacea to modulate immune activity. This hypothesis has been formulated on the basis of preliminary studies showing that ethanolic extracts of these plants, which are high in alkylamides, modulate the activity of lymphocyte and monocyte/macrophage cells in vitro. To test this hypothesis, plant extracts of varying polarity will be prepared from Echinacea purpurea and Spilanthes acmella plant material that has been verified in terms of genus and species. The chemical composition of these extracts will be determined using liquid chromatography coupled to mass spectrometry. Extracts and isolated constituents will be assayed for effects on activity of cells in human lymphocyte and monocyte/macrophage cell lines. Individual compounds will be purified from particularly active extracts, and their activities will be compared to that of complex extracts. Assays using heparinized whole blood will be performed for particularly active extracts and isolated constituents. The results will be analyzed using a sophisticated synergy model to identify specific constituents combinations with immunomodulatory potential. The bulk of these experiments will be carried out in the department of chemistry at UNCG, where undergraduate enrollment consists of 38% minority and 60% women, and undergraduate students are intimately involved in research. This proposal is a multi-disciplinary, collaborative effort between investigators with expertise in immunology and analytical chemistry.